Amylin is a 37-mer aminoacid peptide hormone that is produced in the pancreas and co-secreted with insulin in response to serum glucose levels. Pramlintide is a synthetic analog of amylin (25,28,29tri-L-proline amylin) that retains the biological activity of the hormone while offering superior physical and chemical properties that facilitate drug synthesis and development of a stable drug product for parenteral administration. Pramlintide is approved by US FDA for treating people with type 1 and type 2 diabetes.
Amylin contains a disulphide bridge from Cys-2 to Cys-7 and has an amidated C-terminus. During synthesis amylin is prone to aggregation because of excessive hydrophobic tendencies. This causes low coupling yields, incomplete couplings and side products. The addition of pseudoproline dipeptides to the peptide increases the purity of difficult peptides by decreasing the aggregation.
The synthetic analog pramlintide was arrived at from amylin by substitution of Ala25, Ser28 and Ser29 with proline residues. The pramlintide sequence contains no free carboxyl groups, even at the amidated C-terminus (tyrosine). All the carboxyl groups in pramlintide are amidated, rendering the molecule cationic (protonated histidine, and arginine) at acidic pH. Pramlintide is isolated as a salt with acetate as the counterion. The peptide sequence is:
wherein the line indicates the Cys-Cys disulfide bridge of the bioactive form.
Solid phase peptide synthesis is an alternative to solution phase synthesis. Currently large number of peptides have been successfully produced on a large scale using solid phase peptide synthesis.
Despite having advantages over solution phase synthesis, sometimes the synthesis process becomes highly expensive due to the heavy cost incurred by raw materials used for the synthesis for e.g., protected aminoacids, aminoacid derivatives and specific resins. The use of expensive aminoacid derivatives becomes a major setback in the synthesis, as the process becomes very expensive due to which the commercial scale production of the peptide hinders affecting the commercial business gain.
U.S. Pat. No. 5,686,411 discloses the solid phase synthesis of amylin and amylin analogs using methylbenzhydrylamine anchor-based resin and Nα-Boc/benzyl-side chain protection. Also it discloses the method of treating diabetes mellitus.
U.S. Pat. No. 5,424,394 discloses a solid phase peptide synthesis of amylin or amylin analogs using classical stepwise process wherein such synthetic human amylin is substantially free of deletion and other contaminating peptides having a purity to boat least about 95%. The invention also describes the use of Boc protection and double couplings cycles to get enhanced yield.
As with any drug substance, identifying and quantitating low levels of related substance impurities present in synthesis lots warrants a robust manufacturing process for pramlintide. Missed couplings, double-couplings, and L- to D-aminoacid isomerizations are common errors that occur in peptide synthesis to yield, respectively, single-point aminoacid deletion peptides, addition peptides, and diastereomers as related substances of the desired molecule. Specifically for pramlintide, the 37 aminoacid residue length severely complicates detecting single aminoacid modifications and requires highly selective analytical test methods for purity determinations. Degradation pathways for peptides in acidic aqueous solution frequently involve deamidation at asparagine and glutamine plus hydrolytic backbone cleavage. The 37 aminoacid length and the 8 potential deamidation sites create a potential for many degradation products that differ from pramlintide by modifications at only a single aminoacid. Hence the synthesis of pramlintide poses significant challenge for manufacturers being a difficult peptide to synthesize.
Successful peptide assembly is still hampered by inherent problems such as poor solvation of the growing peptide chain during solid phase synthesis as well as limited solubility of fully protected peptide fragments in the solution approach, often leading to incomplete coupling steps. These undesirable physicochemical problems originate from intermolecular hydrophobic aggregation of the protected peptide chains and/or the formation of secondary structures, most notable β-sheets. Reported attempts to suppress the degenerative effect of such associations during aminoacylation reactions involve essentially “external factors” like solvent composition, elevated temperature, and use of chaotropic salts or solubilizing protecting groups which have been shown to have variable efficiencies. Hydrogen-bonded association has also been prevented by the introduction of an amide protecting group within the peptide chain.
Mutter et al. (1996) were the first to use serine-, threonine-, and cysteine-derived cyclic building blocks (pseudoprolines) to serve as reversible protecting groups for Ser, Thr, and Cys, which have proven to be versatile tools for overcoming some intrinsic problems in the field of peptide chemistry. Mutter et were also the first to report that Ser/Thr-derived oxazolidine and Cys-derived thiazolidine derivatives exert a pronounced effect upon backbone conformation due to their structural similarity with proline itself. Due to the induction of a kink conformation in the peptide backbone, originating in the preference for cis amide bond formation, Ψpro prevent peptide aggregation, self-association, and β-structure formation, thus improving the solvation and coupling kinetics of the growing peptide chain considerably. These building blocks are readily accessible by cyclization Ser, Thr, or Cys with aldehydes or ketones and serve as reversible protecting group in peptide synthesis. As a particular feature, variation of the C-2 substituents directly affects the ring stability, thus allowing for differential chemical stabilities in a variety of synthetic strategies.
Aggregation and hence problems in solid phase synthesis is commonly believed in the art to correlate with occurrence of extended regions of β-sheet structure. A β-sheet structure contents is common to most peptides of at least 10 aminoacids length and do not correlate with any unusual problem in synthetic methodology.
Pseudoproline dipeptides are basically incorporated in the most common peptide synthesis strategies such as a) they can be coupled to growing peptide chains using standard procedures and coupling reagents, b) they are easily cleaved with standard TFA mixtures, and c) they are compatible with synthesis strategies involving Fmoc and Z-aminoacids.
PCT publication WO2009003666 discloses the preparation of Pramlintide via a convergent three fragment synthesis strategy from the fragments comprising the aminoacid residues 1-12, 13-24 and 25-37 respectively. The described procedure uses three pseudoprolines moieties for the synthesis of pramlintide, wherein it improves the solubility of the peptide and prevented or decreased its aggregation.